1. Field of the Invention
This invention relates to sheet materials used for chemical analysis having a multilayer construction. In particular, the present invention relates to test sheets for analyzing blood serum, urine and other biological fluids.
2. Description of the Prior Art
Various types of multilayer sheet materials for chemical analysis are known. They include test sheets for spot analysis of samples in the form of aqueous solutions and, in particular, those of a multilayer construction suited for rapid and simple quantitative analysis and examination of ingredients involved in fluid samples from the living body such as blood, urine, etc. Such test sheets comprise a transparent substrate, one or more layers containing chemical reagents provided thereon and a porour spreading layer superimposed on the chemical reagent layer. These layers are firmly bonded to each other to form a unified structure. In some instances, the substrate may be eliminated.
U.S. Pat. No. 3,992,158 describes multilayer elements for qualitative and quantitative analysis of liquids such as blood serum and urine, which elements preferably comprise an isotropically porous spreading layer in fluid contact with a reagent layer which comprises at least one material interactive with a component or decomposition product of a component of the liquid.
U.S. Pat. No. 3,983,005 describes a multilayer element for analysis of cholesterols in a liquid such as serum. The element is of the type which comprises two superposed layers comprising a spreading layer in liquid contact with a reagent layer, and optionally, a support. Cholesterol oxidase and a cholesterol ester hydrolyzing composition comprising lipase having cholesterol estrase activity and protease are included in the reagent layer. Membrane filters or the like isotropically porous layers can be used as the spreading layer.
In addition, U.S. Pat. No. 4,042,335 describes multilayer elements for analysis of liquids, which comprise three superposed layers including a reagent layer, a radiation-blocking layer and a registration layer. U.S. Pat. No. 4,066,403 describes a multilayer analysis element comprising two reagents and a barrier composition separating the reagents. U.S. Pat. No. 4,050,898 describes a multilayer analysis element having therein a nonionic surfactant-containing spreading layer. U.S. Pat. No. 4,069,016 describes multilayer elements for analysis of a liquid to detect the bilirubin content in the liquid. U.S. Pat. No. 3,526,480 describes integral analysis elements adapted for automated test procedures.
When a drop of the sample fluid to be examined is applied to the spreading layer of such test sheets, diffusion of the fluid into the underlying reagent layer occurs concurrently with lateral spreading in the spreading layer, causing a chemical reaction to proceed upon contact with the chemical reagent in the reagent layer. Since a colored reaction product forms or discoloration takes place, one can quantitatively analyze the extent to which the reaction has proceeded by observing or examining the color of the reagent layer directly or through the substrate. For this purpose, color developing reactions caused by suitable organic reagents in an aqueous medium are particularly preferred. Such analysis self-evidently depends on the reaction between the ingredient tested for involved in the fluid sample and the chemical reagent contained in the reagent layer. Even comparatively rapid reactions take one minute or longer periods while slower reactions take from about 5 to 10 minutes as in the case of reactions relying upon enzymes as an active material at a temperature near 35.degree. C. For instance, Example 3 of British Pat. No. 1,440,464 or French Pat. No. 2,191,734 is an example of an analysis sheet where the reaction time is short. In this Example, the reagent layer is composed of silver chromate and gelatin and upon reaction it turns from reddish brown to yellow due to chlorine ion present in the blood. Example 1 of U.S. Pat. No. 3,983,005 is an example where the reaction time is long. The reagent system used therein is composed of cholesterol oxidase, peroxidase and 4-methoxy-1-naphthol, and when free cholesterol is detected in the serum, the reaction continues over a period of time from about 5 to 20 minutes whereupon the color of the system becomes dark. Further, the existence of ample water is required until termination of the reaction.
To check the effect of the moisture present in the reaction environment in advance of the reaction, a sample fluid was dropped on two separate spots of an analysis sheet. In this case, the analysis sheet was a multilayer analysis element (or sheet) comprising in order a support, a reagent layer and a spreading layer. One of the spots was left open to the air while the other spot was covered with a thin film to prevent evaporation of the moisture after the applied fluid diffused into the spreading layer. In the former spot, the moisture evaporated rapidly thus suppressing the color developing reaction, whereas the latter covered spot remained moist for more than 20 minutes during which time the color developing reaction continued to completion. The above-described comparison indicates that the prevention of moisture escape from the sample fluid applied on the analysis sheet is essential for such a new type of multilayer analysis sheet to be acceptable in the market as a useful tool for simple and rapid quantitative analysis.
If one wants to prevent moisture evaporation from a conventional analysis sheet comprising only a spreading layer, a chemical reagent layer and a substrate, one must apply a cover to the test spot several seconds after the application of the fluid, i.e., after the fluid has diffused into the spreading layer.
Such fluid application followed by the application of a cover seriously conflicts with continuous or automated analysis using an analysis sheet.